8594 Vol.
10, 8594 – 8602, December 15, 2004
Epidermal Growth Factor Receptor (EGFR) Is Overexpressed in
Anaplastic Thyroid Cancer, and the EGFR Inhibitor Gefitinib
Inhibits the Growth of Anaplastic Thyroid Cancer
Bradley A. Schiff,2 Andrea B. McMurphy,2
Samar A. Jasser,1 Maher N. Younes,1 Dao Doan,1
Orhan G. Yigitbasi,1 Seungwon Kim,1 Ge Zhou,1
Mahitosh Mandal,1 Benjamin N. Bekele,3
F. Christopher Holsinger,1 Steven I. Sherman,4
Sai-Ching Yeung,5 Adel K. El-Naggar,1,6 and
Jeffrey N. Myers1,7
Departments of 1Head and Neck Surgery and 2Otorhinolaryngology,
Baylor College of Medicine, Houston, Texas; and Departments of
3
Biostatistics, 4Endocrine Neoplasia and Hormonal Disorders,
5
Ambulatory Treatment and Emergency Care, 6Pathology, and
7
Cancer Biology, The University of Texas M. D. Anderson Cancer
Center, Houston, Texas
ABSTRACT
Purpose: No effective treatment options currently are
available to patients with anaplastic thyroid cancer (ATC),
resulting in high mortality rates. Epidermal growth factor
(EGF) has been shown to play a role in the pathogenesis of
many types of cancer, and its receptor (EGFR) provides an
attractive target for molecular therapy.
Experimental Design: The expression of EGFR was de-
termined in ATC in vitro and in vivo and in human tissue
arrays of ATC. We assessed the potential of the EGFR
inhibitor gefitinib (“Iressa,” ZD1839) to inhibit EGFR acti-
vation in vitro and in vivo, inhibit ATC cellular proliferation,
induce apoptosis, and reduce the growth of ATC cells in vivo
when administered alone and in combination with pacli-
taxel.
Results: EGFR was overexpressed in ATC cell lines in
vitro and in vivo and in human ATC specimens. Activation of
EGFR by EGF was blocked by the addition of gefitinib. In
vitro studies showed that gefitinib greatly inhibited cellular
proliferation and induced apoptosis in ATC cell lines and
Received 4/8/04; revised 9/3/04; accepted 9/13/04.
Grant support: The University of Texas M. D. Anderson Cancer
Center Physician-Scientist Program and Multi-Disciplinary Research
Program in Thyroid Cancer and by The Golfers Against Cancer. Ge-
fitinib was provided as a gift from AstraZeneca.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Jeffrey N. Myers, Department of Head and Neck
Surgery, Unit 441, The University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-
745-2667; Fax: 713-794-4662; E-mail: jmyers@mdanderson.org.
©2004 American Association for Cancer Research.
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Research.
Clinical Cancer Research
slowed tumor growth in a nude mouse model of thyroid
carcinoma cells injected subcutaneously.
Conclusions: ATC cells consistently overexpress EGFR,
rendering this receptor a potential target for molecular
therapy. Gefitinib effectively blocks activation of EGFR by
EGF, inhibits ATC cellular proliferation, and induces apo-
ptosis in vitro. Our in vivo results show that gefitinib has
significant antitumor activity against ATC in a subcutane-
ous nude mouse tumor model and therefore is a potential
candidate for human clinical trials.
INTRODUCTION
The annual prevalence of thyroid cancer in the United
States was expected to be ⬃22,000 people in 2003 (1). The vast
majority of thyroid carcinomas are differentiated and can often
be cured surgically. Anaplastic thyroid carcinomas are relatively
rare, constituting only 1.6% all of the thyroid cancers (2), and
show extremely aggressive behavior, leading to a high mortality
rate. Patients with anaplastic thyroid cancer (ATC) face a uni-
formly dismal prognosis, with average 5-year survival rates of
⬍10% and a median survival time of 3 months (3). No effective
treatment options currently are available to patients with ATC.
Targeted molecular therapy offers potential treatment al-
ternatives for patients with ATC. Epidermal growth factor
(EGF) and its receptor (EGFR) have been implicated in the
pathogenesis of many different types of cancer and thus provide
attractive targets for molecular therapy. EGFR, which is en-
coded by the c-erb proto-oncogene, is a Mr 170,000 transmem-
brane cell-surface glycoprotein consisting of an extracellular
ligand binding domain, a transmembrane domain, and an intra-
cellular domain with intrinsic tyrosine kinase activity (4, 5).
Dimerization of EGFR following the binding of the ligand
results in trans-phosphorylation of this receptor and subsequent
activation of several downstream signal transduction pathways,
including the mitogen-activated protein kinase and phosphati-
dylinositol-3⬘ kinase signaling pathways, which are involved in
promoting cellular proliferation and survival (6 – 8). In preclin-
ical studies, EGF has been shown to stimulate follicular cell
proliferation and to enhance the migration and invasiveness of
papillary thyroid cancer (9 –11). The medical literature gener-
ally supports the concept that EGFR, although expressed at
higher levels in anaplastic and papillary cancers than in normal
thyroid tissue, is present in all of the thyroid tissues, but the data
in different studies often vary widely (9, 12–17). In an in vitro
study by Bergstrom et al. (18), EGFR was expressed in all six
ATC cell lines examined, and constitutive phosphorylation of
EGFR was found in three of the six cell lines tested.
High expression of EGFR appears to be a negative prog-
nostic factor in multiple types of tumors, including breast cancer
(19) and bladder cancer (20); however, few studies have exam-
ined the clinical implications of EGFR expression and location

in thyroid cancer. A recent study found a statistically significant
correlation between the staining intensity of EGF and recurrence
of papillary thyroid cancer (16). In another report, cytoplasmic
immunopositivity was significantly associated with the extent of
primary tumor infiltration in papillary thyroid cancer, whereas
membranous staining was not. Furthermore, in a multivariate
survival analysis, strong cytoplasmic EGFR staining of papillary
thyroid cancer was significantly associated with a decrease in
recurrence-free survival (21). These findings suggest that the
molecular blockage of EGFR activation has the potential to
reduce thyroid tumor growth, making EGFR an attractive target
for molecular therapy against ATC.
Gefitinib (“Iressa,” ZD1839; AstraZeneca, Wilmington,
DE), a synthetic anilinoquinazoline, is an orally active EGFR
inhibitor that is highly selective, with minimal activity against
other tyrosine kinases. Gefitinib has been shown to block EGF-
stimulated EGFR autophosphorylation (22) and EGFR-medi-
ated downstream signal transduction. The drug also has shown
activity against a variety of human tumor cells in vitro and
additive to synergistic effects when combined with radiation
therapy or chemotherapy (23, 24). Results of Phase I trials have
indicated that this agent offers good bioavailability and tolera-
bility (25). Many Phase II and III studies currently are underway
to assess the effectiveness of gefitinib against a variety of
carcinomas, including breast, lung, colorectal, head and neck,
prostate, and renal. Two recent studies have suggested that
response to gefitinib therapy is linked to mutations in the EGFR
gene (26, 27). Although numerous studies already have shown
that gefitinib possesses clinically meaningful antitumor activity
in several malignancies (28), to our knowledge, no clinical trials
yet exist to determine the effectiveness of gefitinib against ATC.
Thus, we investigated the role of EGFR and its inhibitor ge-
fitinib in ATC to determine whether gefitinib possesses mean-
ingful antitumor activity against ATC, potentially justifying
clinical trials.
MATERIALS AND METHODS
Animals. Male athymic nude mice, ages 8 to 12 weeks,
were purchased from the animal production area of the National
Cancer Institute Frederick Cancer Research and Development
Center (Frederick, MD). The mice were housed and maintained
in laminar flow cabinets under specific pathogen-free conditions
in facilities approved by the American Association for Accred-
itation of Laboratory Animal Care in accordance with current
regulations and standards of the United States Department of
Agriculture, the United States Department of Health and Human
Services, and the NIH. The mice were treated in accordance
with the Animal Care and Use Guidelines of the University of
Texas M. D. Anderson Cancer Center (Houston, TX) under a
protocol approved by the Institutional Animal Care Use Com-
mittee.
Cell Lines and Culture Conditions. The papillary thy-
roid carcinoma cell line NPA187 and the ATC cell lines KAT-4,
K18, C643, HTH, ARO, and DRO were used. Oral cavity
squamous cell cancer line TU167 was used as a positive control,
and the fibroblast line 3T3 was used as a negative control. The
cells were grown in Dulbecco’s modified Eagle’s medium sup-
plemented with 10% fetal bovine serum (DMEM-10% FBS),
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Clinical Cancer Research 8595
L-glutamine, penicillin, sodium pyruvate, nonessential amino
acids, and a twofold vitamin solution (Life Technologies, Inc.,
Rockville, MD). Adherent monolayer cultures were maintained
on plastic and incubated at 37°C in 5% carbon dioxide and 95%
air. The cultures were free of Mycoplasma species. All of the
cell lines injected into the mice tested free of the following
pathogenic murine viruses: reovirus type 3, pneumonia virus, K
virus, Theiler’s encephalitis virus, Sendai virus, minute virus,
ectromelia virus, and lactate dehydrogenase virus (as assayed by
M.A. Bioproducts, Walkersville, MD). The cultures were main-
tained no longer than 12 weeks after recovery from frozen
stocks.
Reagents. For in vitro administration, gefitinib was dis-
solved in dimethyl sulfoxide to a concentration of 20 mmol/L.
For in vivo testing, gefitinib was dissolved in a lactate salt
solution. Propidium iodide (PI) and tetrazolium (MTT) were
purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Western Immunoblot Analysis. All of the cells were
incubated in serum-free medium for 24 hours. Cells were incu-
bated with gefitinib for 1 hour at concentrations ranging from
0.01 to 100 ␮mol/L before addition of EGF (40 ng/mL) for 15
minutes. The cells then were washed with PBS, and lysis buffer
was added [1% Triton X-100, 20 mmol/L Tris (pH 8.0), 137
mmol/L sodium chloride, 10% glycerol (v/v), 2 mmol/L EDTA,
1 mmol/L phenylmethylsulfonyl fluoride, 20 ␮mol/L aprotinin-
leupeptin-trypsin inhibitor, and 2 mmol/L sodium orthovana-
date]. The cells were scraped and spun in a centrifuge to remove
insoluble proteins. The samples were diluted in sample buffer
[10% SDS, 0.5 mmol/L Tris-HCl (pH 6.8), 1 mol/L dithiothre-
itol, 10% (v/v) glycerol, and 1% bromphenol blue] and boiled.
The proteins (50 ␮g) were resolved on polyacrylamide gel
electrophoresis and electrophoretically transferred onto 0.45-␮g
nitrocellulose membranes. The membranes were blocked with
5% (w/v) nonfat milk in 0.1% Tween 20 (v/v) in Tris-buffered
saline, probed with rabbit monoclonal anti-EGFR (1:2000; Up-
state Biotechnology, Inc., Lake Placid, NY) in 1% nonfat milk,
and incubated with peroxidase-conjugated sheep antirabbit im-
munoglobulin (1:2000; Amersham, Piscataway, NJ) in 1% non-
fat milk. The blots also were probed with rabbit anti–phospho-
EGFR (p-EGFR 1068; Cell Signaling Technologies, Beverly,
MA), diluted 1:1000 in 1% nonfat milk, and incubated with
peroxidase-conjugated sheep antirabbit IgG (1:3000). All of the
blots were probed with anti–␤-actin (1:1000) in 1% nonfat milk,
followed by horseradish peroxidase– conjugated donkey antirab-
bit IgG (1:2000; Amersham) in 1% nonfat milk. Protein bands
were visualized using an enhanced chemiluminescence detec-
tion system (Amersham).
ELISA. Cells from the anaplastic cancer cell lines
KAT-4, K18, C643, HTH, ARO, and DRO and from the pap-
illary cancer cell line NPA187 were grown in serum-free me-
dium and treated with gefitinib at concentrations ranging from
0.01 to 100 ␮mol/L. After 24 hours, the supernatant was col-
lected, and the cells were counted. The supernatants were ex-
amined for transforming growth factor ␣ (TGF-␣) and EGF. All
of the kits were purchased from R&D Systems (Minneapolis,
MN), and all of the experiments were performed according to
the manufacturer’s instructions. The absorbance and concentra-
tion (pg/mL) were measured using a microplate reader at 450
8596 EGFR Inhibitor Gefitinib Inhibits ATC
nm. For each cell line, the results were plotted as the ratio of the
concentration to the total number of cells.
PCR Analysis of EGFR Mutations. The PCR was used
to amplify exons 18, 19, and 21 from the EGFR gene using
genomic DNA isolated from the following tumor-derived cell
lines: papillary thyroid cancer cell line NPA187 and ATC cell
lines DRO, K18, ARO, HTH-74, C643, and KAT-4. PCR am-
plicons were purified using QIAquick PCR purification kit
(Qiagen, Valencia CA) and then sent to LoneStar Labs (Hous-
ton, TX) for sequencing. Electropherograms were analyzed for
the presence of mutations.
Measurement of Cell Proliferation. Gefitinib was
tested on the KAT-4 ATC cell line using an MTT-based assay,
which measures cell proliferation based on the ability of live
cells to convert MTT to dark blue formazan. Two thousand cells
were grown in DMEM-10% FBS, with and without 0.01 to 100
␮mol/L gefitinib, in 96-well tissue culture plates. After 24
hours, the cells again were incubated with medium alone or
medium containing various concentrations of gefitinib diluted in
dimethyl sulfoxide. After a 3-day incubation, the number of
metabolically active cells was determined by MTT assay using
a 96-well microtiter plate reader (MR-5000; Dynatech Labora-
tories Inc., Chantilly, VA) at an absorbance of 570 nm. The
absorbance for a control plate, which was not seeded with any
cells during initial plating, was subtracted from the absorbance
of every other well.
Measurement of Cell Death. Cells were plated at a
density of 5 ⫻ 105 cells in 38 mm2 six-well plates (Costar,
Cambridge, MA) and maintained for 24 hours before treatment
with gefitinib. After 24 hours, gefitinib was added at various
concentrations. After treatment for 48 hours, PI staining of
hypodiploid DNA determined the extent of cell death. A total of
3 ⫻ 105 cells were resuspended in a Nicoletti buffer [50 ␮g/mL
PI, 0.1% sodium citrate, 0.1% Triton X-100, and 1 mg/mL
RNase (Roche, Basel, Switzerland) in PBS] for 20 minutes at
4°C. Cells then were analyzed by flow cytometry, and the
sub-G0/G1 fraction was measured using the Lysys software
(Becton Dickinson, Franklin Lakes, NJ).
Immunohistochemical Analysis of Murine Tumor Fro-
zen Sections and Human Tumor Tissue Arrays. Immuno-
histochemical analysis was performed using rabbit anti-EGFR
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Frozen
tumors were sectioned (8 to 10 ␮m thick), mounted on posi-
tively charged Superfrost slides (Fisher Scientific, Houston,
TX), air dried for 30 minutes, and fixed in cold acetone for 10
minutes. The slides were washed three times with PBS (pH 7.5),
blocked for 20 minutes at room temperature in PBS supple-
mented with 1% normal goat serum and 5% normal horse serum
(protein-blocking solution), and incubated with primary anti-
body for 18 hours at 4°C. Samples then were washed three times
for 3 minutes, blocked with protein-blocking solution for 10
minutes, and incubated with AlexaFluor 594 – conjugated goat
antirabbit IgG (Molecular Probes, Eugene, OR) for 1 hour at
room temperature in the dark. Samples again were washed three
times for 5 minutes and then counterstained with 300 ␮g/mL
Hoechst stain for 1 to 2 minutes at room temperature. The slides
were washed again with PBS and then mounted using propyl
gallate.
Immunofluorescence microscopy was performed in a Ni-
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kon Microphot-FXA (Nikon Inc., Tokyo, Japan) equipped with
an HBO 100 mercury lamp and narrow bandpass filters to
individually select for green, red, and blue fluorescence
(Chroma Technology Corp., Brattleboro, VT). Images were
captured using a cooled charge-coupled device Hamamatsu
5810 camera (Hamamatsu Corp., Bridgewater, NJ) and Optimas
Image Analysis software (Media Cybernetics, Silver Spring,
MD). Photomontages were prepared using Adobe Photoshop
software (Adobe Systems Inc., San Jose, CA).
Human Thyroid Tissue Arrays. Thyroid tumor tissue
arrays representative of the entire spectrum of benign and ma-
lignant neoplasms, including ATC constructed at the head and
neck tissue care facility, were used to screen for EGFR expres-
sion. The arrays represented 14 papillary carcinomas, 6 anaplas-
tic carcinomas, and 9 samples of nondiseased thyroid tissue.
Two cores of each sample were placed differentially in the
recipient block. Two pathologists scored these blindly and in-
dependently on a scale of 0 to 3. Whenever a discrepancy in
scoring was noted, both pathologists reexamined the sample in
question, and a consensus was reached.
In vivo Xenografts. Tumor response to gefitinib was
gauged using a nude mouse model of thyroid cancer. KAT-4
ATC cells were harvested from subconfluent cultures by
trypsinization and then washed. Using a 30-gauge needle under
direct visualization, 5 ⫻ 106 KAT-4 cells diluted in 30 ␮L of
serum-free medium were injected subcutaneously into the cer-
vical area. Tumors were allowed to grow for 14 days; all of the
mice then were weighed, and all of the tumors were measured
using microcalipers.
Tumor volume was calculated using the formula
(A)(B2)␲/6, where A was the length of the longest aspect of the
tumor, and B was the length of the tumor perpendicular to A.
The two mice with the largest tumors and the three with the
smallest tumors were excluded from the analysis. All of the
other mice then were placed into groups of five mice with
similar average tumor volumes. The groups then were random-
ized into seven treatment groups. Each mouse in group 1, the
control group, received sodium lactate at a dosage of 0.2 mL/d
for 5 days (Monday through Friday) for 2 weeks via oral gavage.
All of the other groups received gefitinib dissolved in 0.2 mL of
sodium lactate solution via oral gavage. The mice in group 2
each received 0.9 mg/d (30 mg/kg) for 5 days per week for 2
weeks; mice in group 3, 1.8 mg/d (60 mg/kg) for 5 days per
week for 2 weeks; mice in group 4, 2.7 mg/d (90 mg/kg) for 5
days per week for 2 weeks; mice in group 5, 4.5 mg/d (150
mg/kg) for 5 days per week for 2 weeks; mice in group 6, 4.5
mg/d (150 mg/kg) for 5 days per week for 2 weeks ⫹ paclitaxel
(200 ␮g/wk injected intraperitoneally); and mice in group 7, 4.5
mg/d (150 mg/kg) every other day (QOD), Monday, Wednes-
day, and Friday for 2 weeks. The mice were weighed and the
tumors were measured on days 8 and 12 using microcalipers
until the mice were euthanatized after 2 weeks of treatment.
After the mice were euthanatized, the tumors again were meas-
ured, and the mice were weighed.
For immunohistochemical and routine hematoxylin and
eosin staining, one part of the tissue was fixed in formalin and
embedded in paraffin, and another part was embedded in opti-
mal cutting-temperature compound (Miles Inc., Elkhart, IN),

Table 1 Level of staining for EGFR in normal, papillary, and
anaplastic thyroid tissue from 29 human subjects
EGFR staining
Level of staining Percentage
Tissue No. of of specimen
type subjects 0 1 2 3 staining
Normal 9 9 0 0 0 0
Papillary 14 12 2 0 0 14
Anaplastic 6 1 3 2 0 83
Abbreviation: EGFR, epidermal growth factor receptor.
rapidly frozen in liquid nitrogen, and stored at ⫺80°C. Hema-
toxylin and eosin staining confirmed the presence of tumors.
Statistical Analyses. To examine the results of EGFR in
human thyroid tissue arrays, we performed three pairwise com-
parisons using Fisher’s exact test. In the first analysis, the type
I error rate was controlled at 0.017, guaranteeing that the overall
type I error rate would be controlled at 0.05. To determine the
IC50 for MTT and apoptosis assays, linear interpolation was
used. We chose this method because the changes in absorbance
as a percentage of the control and in apoptosis were linearly
related to gefitinib levels in the range of the IC50.
Two methods were used to analyze the data from the study
involving the model in which nude mice were injected subcu-
taneously. The purpose of this analysis was to determine
whether there was a statistically significant difference in change
in tumor size between mice treated with gefitinib at dosages of
30, 60, 90, and 150 mg/d, 150 mg/d plus paclitaxel, and the
control group. The endpoint of interest was the percentage
change from initial tumor size at day 12 of treatment. The
Wilcoxon rank sum test was used to detect statistical signifi-
cance in the percentage change in tumor size from the initial
tumor size at day 12. The Jonckheere-Terpstra test was used to
test for evidence of a trend of decreasing tumor size with
increasing treatment dosage.
Fig. 1 Representative samples of the tissue arrays for normal thyroid tissue, papillary thyroid cancer, and ATC after staining for EGFR. Staining
levels were 0, 1, and 2, respectively.
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Clinical Cancer Research 8597
RESULTS
Production of Growth Factors EGF and TGF-␣ by
Thyroid Cancer Cell Lines. To assess whether ATC cells
produce the EGFR ligands EGF and TGF-␣, ELISA was used to
assess their levels in culture supernatants from six anaplastic
and one papillary cancer cell lines. EGF was not present at an
appreciable level in the supernatants from any of the cell lines
examined. TGF-␣ was present at a level of 38.5 pg/mL per cell
number in the medium from the ATC cell line DRO but was not
present at an appreciable level in any of the other cell lines
examined.
Expression of EGFR in Human Tissue Arrays. To
determine the expression level of EGFR in normal and neoplas-
tic human thyroid tissue, tissue arrays of surgical specimens
composed of normal thyroid tissue, papillary thyroid cancer, and
ATC were obtained from Dr. Adel El-Naggar (Department of
Surgical Pathology, M. D. Anderson Cancer Center). None of
the nine normal tissue specimens stained positive for EGFR. Of
the specimens from the 14 patients with papillary thyroid can-
cer, 2 stained positive for EGFR. Specimens from five of the six
patients with ATC stained positive for EGFR; specimens from
two of these five patients stained at level 2, whereas specimens
from the three other patients stained at level 1. Comparisons of
ATC specimens with normal thyroid tissue specimens and ATC
specimens with papillary thyroid cancer specimens revealed
statistically significant differences in EGFR expression (P ⫽
0.002 and P ⫽ 0.007, respectively). No statistically significant
difference in EGFR expression was found between the papillary
thyroid cancer and the normal thyroid tissue specimens (P ⫽
0.5; Table 1, Fig. 1).
Expression of EGFR/Phospho-EGFR in Human Thy-
roid Cancer Cell Lines. To assess expression of EGFR and
p-EGFR in human thyroid tissue cell lines in vitro, Western blot
analysis was performed on cellular lysates of the six ATC cell
lines (ARO, C643, DRO, HTH, K18, and KAT-4) and the
papillary cell line (NPA187). Five of the six ATC cell lines (all
8598 EGFR Inhibitor Gefitinib Inhibits ATC
Fig. 2 Western blot analysis of cellular lysates of six ATC cell lines
(ARO, C643, DRO, K18, and KAT-4) and the papillary cell line
NPA187. Five of the six ATC cell lines (all except DRO) stained
positive for EGFR, whereas NPA187 was negative for EGFR.
except DRO) stained positive for EGFR (Fig. 2), and the pap-
illary cell line, NPA187, was negative for EGFR. In additional
Western blot analyses, all of the cell lines were negative for the
activated, or phosphorylated, form of EGFR (p-EGFR) unless
stimulated with EGF (data not shown).
In vivo Expression of EGF, EGFR, and p-EGFR. A
nude mouse model was used to determine the expression of
EGF, EGFR, and p-EGFR in mice implanted subcutaneously
with 2.5 ⫻ 106 cells of KAT-4 ATC. Tumors were allowed to
form, and after 1 month, the mice were euthanatized, and the
tumors were subjected to immunohistochemical staining. Nor-
mal murine thyroid tissue and ATC implants expressed EGFR
and p-EGFR.
PCR Analysis of EGFR Showed No Mutations. Anal-
ysis of exons 18, 19, and 21 of EGFR showed no mutations in
the six ATC cell lines tested (DRO, K18, ARO, HTH-74, C643,
and KAT-4) or the papillary cell line NPA187.
Gefitinib Inhibited EGFR Phosphorylation in KAT-4
Cells In vitro. To assess whether gefitinib could inhibit EGFR
signaling in ATC in vivo, KAT-4 ATC cell lines were exposed
to serum-free medium for 24 hours. The cells then were treated
with various concentrations of gefitinib in serum-free medium
for 1 hour. The level of gefitinib ranged from 0.01 to 100
␮mol/L. After 1 hour of exposure, recombinant EGF at a dosage
of 40 ng/mL was added for 15 minutes. In Western blot analysis,
the KAT-4 cells showed a high level of p-EGFR when stimu-
lated with EGF. Gefitinib partially blocked EGF autophospho-
rylation of EGFR at a concentration of 0.01 ␮mol/L and almost
completely blocked autophosphorylation at 1 ␮mol/L. At a
concentration of 10 ␮mol/L, gefitinib completely blocked
EGFR autophosphorylation (Fig. 3).
Gefitinib Inhibited EGFR Phosphorylation in KAT-4
Cells In vivo. To assess the efficacy of gefitinib inhibition of
autophosphorylation of EGFR in ATC cells in vivo, mice im-
planted subcutaneously with the KAT-4 ATC cell line, which
expresses a high level of p-EGFR, were treated with gefitinib at
dosages of 30, 60, 90, and 150 mg/kg/d and at a dosage of 150
mg/kg/QOD. The mice treated with gefitinib at 30 and 60
mg/kg/d showed high levels of p-EGFR. The tumor sections
from the mice treated with gefitinib at 90 mg/kg/d showed high
levels of p-EGFR but lower expression than in tumor sections
from the mice treated with 30 or 60 mg/kg/d. The mice treated
with gefitinib at 150 mg/kg/QOD showed p-EGFR expression if
they were euthanatized immediately before the final treatment
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but did not express p-EGFR if euthanatized 6 hours after the
final treatment. The mice treated with gefitinib at a dosage of
150 mg/kg/d showed no expression of p-EGFR, whether eu-
thanatized either immediately before or 6 hours after the final
treatment (Fig. 4).
Gefitinib Inhibited Cellular Proliferation of KAT-4
Cells In vitro. To determine the effect of inhibition of EGFR
signaling on ATC cell proliferation in vitro, three MTT assays
were performed using gefitinib at concentrations ranging from 1
to 20 ␮mol/L (Fig. 5). The lowest concentration at which
cellular proliferation was inhibited was 6 ␮mol/L. The IC50 was
8.36 ␮mol/L, and maximal inhibition occurred at a concentra-
tion of 14 ␮mol/L.
Gefitinib Induced Apoptosis in KAT-4 Cells In vitro.
To assess the effects of gefitinib on induction of cell death of
ATC cells, we performed a PI apoptosis assay on KAT-4 cells
after 48 hours of treatment. When treated at a concentration of
8 ␮mol/L (the IC50 for the MTT assays), 5.5% of the cells
underwent apoptosis. At a concentration of 12 ␮mol/L, 27.6%
of the cells were apoptotic; at 19 ␮mol/L, 54.7%; at 22 ␮mol/L,
80.7%; and at 50 ␮mol/L, 94.5% (Fig. 6). The estimated IC50
for the apoptosis assays was 18.35 ␮mol/L.
Gefitinib Alone and in Combination with Paclitaxel
Reduced KAT-4 Tumor Size and Growth in a Nude Mouse
Model of Thyroid Cancer. A nude mouse model of thyroid
cancer was used for the in vivo portion of the study to assess the
in vivo antitumor activity of gefitinib. At day 12, the control
group of mice showed an increased tumor size of 173% of the
initial tumor size. The groups treated with gefitinib at dosages of
30, 60, 90, and 150 mg/kg/d had increased tumor sizes at day 12
of 158%, 165%, 112%, and 104% of the initial tumor size,
respectively. The group treated with paclitaxel alone had an
increase in tumor size at day 12 of 134% of initial tumor size.
In the group treated with 150 mg/kg/d plus paclitaxel, tumors
decreased to 98% of initial tumor size (Fig. 7).
DISCUSSION
Because patients with ATC face a dismal prognosis and
have no effective treatment options, novel treatments are
needed. The EGFR is involved in the pathogenesis or progres-
sion of many different types of cancers and therefore is a
potential target for molecular therapy in ATC. However, little
Fig. 3 Western blot analysis of KAT-4 ATC cell lines treated with
various concentrations of gefitinib and subsequently exposed to EGF.
Gefitinib partially blocked EGF autophosphorylation of EGFR at a
concentration of 0.01 ␮mol/L and almost completely blocked autophos-
phorylation at 1 ␮mol/L. At a concentration of 10 ␮mol/L, gefitinib
completely blocked EGFR autophosphorylation.
 Clinical Cancer Research 8599

Fig. 4 Immunohistochemical results of mice subcutaneously implanted with KAT-4 ATC cell line and treated with various dosages of gefitinib. The
tumors of mice treated with 30 and 60 mg/kg QD (QD, every day) of gefitinib showed high levels of p-EGFR, whereas the tumors of mice treated
with 90 mg/kg/d showed lower expression of p-EGFR. The tumors of mice treated with 150 mg/kg QOD (QOD, every other day) showed p-EGFR
expression if the mice were euthanatized immediately before the final treatment with gefitinib (48 hours after the previous gefitinib treatment) but
did not express p-EGFR if sacrificed 6 hours after the final treatment. The tumors of mice treated with 150 mg/kg/d of gefitinib showed no expression
of p-EGFR whether the mice were euthanatized either immediately before or 6 hours after the final treatment with gefitinib. This showed that gefitinib
at a dosage of 150 mg/kg/d can completely block expression of p-EGFR in a nude mouse model of thyroid carcinoma. * Killed immediately before
treatment. ** Killed 6 hours after treatment.

research has been done to examine the role of EGFR in ATC

and the effectiveness of anti-EGFR therapies against ATC.
Gefitinib is an EGFR tyrosine kinase inhibitor that has already
been shown to have a favorable safety and tolerability profile in
numerous Phase I clinical trials and therefore is a promising area
of investigation in the search for effective treatments for patients
with ATC.
To initiate studies, we performed ELISA to determine the
expression of growth factors TGF-␣ and EGF. EGF was not
present in any of the six ATC cell lines examined, and TGF-␣
was present only in the DRO cell line. DRO, the only ATC cell
line that did not express EGFR, also was the only tested cell line
that expressed TGF-␣. In papillary thyroid cancer, TGF-␣ and
EGFR expression often are linked.
Our thyroid tissue arrays showed significantly greater
EGFR expression in ATC specimens than in either normal
thyroid tissue or papillary thyroid cancer specimens (P ⫽ 0.002
and 0.007, respectively). The medical literature on this topic is
mixed. Some studies (29) report that EGFR is expressed only in
diseased thyroid tissue, whereas others report that EGFR is
expressed in all of the thyroid tissues (9). However, most studies
suggest that EGFR is expressed at a higher level in thyroid
cancer than in normal thyroid tissue (30, 31). The data from our
tissue arrays support this finding and suggest that EGFR is
highly expressed in ATC. Fig. 5 MTT assay of KAT-4 ATC cells exposed to various concentra-
tions of gefitinib. The lowest concentration at which cellular prolifera-
Our analysis of the expression of EGFR by Western blot
tion was inhibited was 6 ␮mol/L, and the IC50 was 8.36 ␮mol/L.
analysis showed no expression of EGFR in the papillary cell line Maximal inhibition occurred at a concentration of 14 ␮mol/L. Thus,
NPA187 but did show EGFR expression in five of the six ATC gefitinib is able to effectively inhibit ATC cellular proliferation.

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8600 EGFR Inhibitor Gefitinib Inhibits ATC
cell lines. The results also showed that EGFR was not consti-
tutively phosphorylated in any of the ATC cell lines tested, but
EGFR phosphorylation was readily observed in these cell lines
after stimulation with EGF. These data are in concordance with
those from previous studies that suggest that EGFR is expressed
in ATC cell lines, but they contradict the literature that suggests
that EGFR is constitutively activated in some ATC cell lines.
However, immunohistochemical staining of sections of subcu-
taneously implanted KAT-4 tumors revealed that those tumors
constitutively expressed EGFR and p-EGFR, as did normal
murine thyroid tissue, suggesting that EGFR activation is up-
regulated in vivo.
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Research.
Fig. 6 PI apoptosis assay on KAT-4 cells after 48
hours of treatment with gefitinib. When treated at
a concentration of 8 ␮mol/L (the IC50 for the MTT
assays), 5.5% of the cells underwent apoptosis.
The estimated IC50 for the apoptosis assays was
18.4 ␮mol/L. At a gefitinib concentration of 22
␮mol/L, 80.7% of cells underwent apoptosis,
whereas at 50 ␮mol/L, 94.5% of cells underwent
apoptosis. This shows that gefitinib is able to
induce apoptosis in ATC cell lines.
We also showed that gefitinib blocked EGF-mediated ac-
tivation of EGFR on ATC cell lines in vitro. The mice treated
with 30 and 60 mg/kg/d of gefitinib showed high levels of
p-EGFR. Specimens from the mice treated with 90 mg/kg/d of
gefitinib showed high levels of p-EGFR but did not stain as
positively as did those from the mice treated with 30 or 60
mg/kg/d. The mice treated with gefitinib 150 mg/kg/d showed
no expression of p-EGFR regardless of whether they were
euthanatized 6 hours after the final treatment or immediately
before the final treatment was scheduled, a fact that indicates
that gefitinib at a dosage of 150 mg/kg/d is able to continuously
suppress EGFR phosphorylation. The mice treated with 150
Fig. 7 A nude mouse model of thyroid
carcinoma cells injected subcutaneously
was used to assess the in vivo antitumor
activity of gefitinib. At day 12, the control
group of mice showed an increased tumor
size of 173% of the initial tumor size. The
groups treated with gefitinib at dosages of
30, 60, 90, and 150 mg/kg/d had increased
tumor sizes at day 12 of 158%, 165%,
112%, and 104% of the initial tumor size,
respectively. The group treated with pacli-
taxel alone had an increase in tumor size at
day 12 of 134% of initial tumor size. In the
group treated daily with 150 mg/kg/d ge-
fitinib ⫹ paclitaxel, tumors decreased to
98% of initial tumor size. This shows that
gefitinib, alone and in combination with
paclitaxel, was able to reduce the growth
of ATC in a nude mouse model of thyroid
carcinoma cells injected subcutaneously.

mg/kg/QOD of gefitinib showed p-EGFR expression if euthana-
tized immediately before the final treatment was supposed to be
given but did not express p-EGFR if euthanatized 6 hours after
the final treatment, showing that a dosage of 150 mg/kg is
unable to suppress EGFR phosphorylation for 48 hours. Our
data show that gefitinib at a daily dosage of 150 mg/kg is able
to suppress EGFR activation for 24 to 48 hours in a nude mouse
model of thyroid cancer.
After establishing that EGFR is overexpressed in ATC and
that gefitinib can suppress EGFR phosphorylation in vitro and in
vivo, we then examined the effect of gefitinib on ATC cell
growth and death. An MTT assay showed that 12 ␮mol/L of
gefitinib caused near-total growth inhibition. A PI apoptosis
assay revealed that 22 ␮mol/L of gefitinib induced a rate of
apoptosis ⬎80%. Thus, gefitinib is able to effectively inhibit
ATC cellular proliferation and induce apoptosis in ATC cell
lines, providing further evidence that gefitinib is effective
against ATC.
The final portion of our experiments used a nude mouse
model to examine the effects of various dosing schedules of
gefitinib alone or in combination with paclitaxel on subcutane-
ously implanted KAT-4 tumors. The difference in change in
tumor size between the control group and the group treated with
gefitinib at a dosage of 150 mg/d ⫹ paclitaxel approached but
did not achieve statistical significance (P ⫽ 0.06). However, if
a trend analysis is done looking at increasing doses of gefitinib
(and including gefitinib 150 mg/d ⫹ paclitaxel as the group
receiving the highest level of treatment), there is a significant
difference between change in tumor size and increasing treat-
ment dosage (P ⫽ 0.015).
In summary, EGF has been shown to play a role in the
pathogenesis of many types of cancer, and its receptor provides
a promising target for molecular therapy. Little research has
been done to examine the role of EGFR in ATC. Novel treat-
ment strategies are needed for patients with ATC because cur-
rent treatment options offer little benefit. We showed that EGFR
is increased in ATC cell lines in vitro and in vivo and in human
ATCs. Gefitinib was able to inhibit EGFR phosphorylation in
vitro and in vivo and to reduce cellular proliferation and induce
apoptosis in ATC cell lines. Finally, gefitinib alone and in
combination with paclitaxel was able to reduce the growth of
ATC cells in a nude mouse model of thyroid carcinoma cells
injected subcutaneously. The favorable safety profile of ge-
fitinib has already been proven in Phase I clinical trials, and
based on the data presented here, clinical trials of gefitinib in
patients with ATC are warranted.
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 Editor's Note Clinical
Cancer
Research
Editor's Note: Epidermal Growth Factor
Receptor (EGFR) Is Overexpressed in
Anaplastic Thyroid Cancer, and the EGFR
Inhibitor Gefitinib Inhibits the Growth of
Anaplastic Thyroid Cancer
Bradley A. Schiff, Andrea B. McMurphy, Samar A. Jasser,
Maher N.Younes, Dao Doan, Orhan G.Yigitbasi, Seungwon Kim,
Ge Zhou, Mahitosh Mandal, Benjamin N. Bekele,
F. Christopher Holsinger, Steven I. Sherman, Sai-Ching Yeung,
Adel K. El-Naggar, and Jeffrey N. Myers

The editors are publishing this note to alert readers to a concern about this article (1):
in Fig. 2B, a splice line is missing in the NPA 187 lane of the b-actin panel.

Reference
1. Schiff BA, McMurphy AB, Jasser SA, Younes MN, Doan D, Yigitbasi OG, et al. Epidermal growth
factor receptor (EGFR) is overexpressed in anaplastic thyroid cancer, and the EGFR inhibitor
gefitinib inhibits the growth of anaplastic thyroid cancer. Clin Cancer Res 2004;10:8594–602.

Published first August 1, 2019.

Clin Cancer Res 2019;25:4862
doi: 10.1158/1078-0432.CCR-19-2052
2019 American Association for Cancer Research.

4862 Clin Cancer Res; 25(15) August 1, 2019

Epidermal Growth Factor Receptor (EGFR) Is Overexpressed
in Anaplastic Thyroid Cancer, and the EGFR Inhibitor
Gefitinib Inhibits the Growth of Anaplastic Thyroid Cancer
Bradley A. Schiff, Andrea B. McMurphy, Samar A. Jasser, et al.

Clin Cancer Res 2004;10:8594-8602.

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